remant bahadur k.c., bindu thapa and narayan bhattarai

DOI 10.1515/ntrev-2013-0026 Nanotechnol Rev 2014; 3(3): 269–280

Review

Remant Bahadur K.C., Bindu Thapa and Narayan Bhattarai*

Gold nanoparticle-based gene delivery: promises and challenges

Abstract: Gold nanoparticles have emerged as a promising material for biomedical research due to ease of synthesis and highly adjustable optical properties, which can be utilized in the imaging of different diseases. Gold nano-particles are fabricated by grafting biocompatible poly-mers and natural or synthethic biomolecules and present a novel avenue for engineering multifunctional smart systems. Many reports on the significant achievements and the bioconjugation chemistry promise to expand the application spectrum of gold nanoparticles. This review summarizes the current state-of-the-art development of functionalized gold nanoparticles for cancer gene therapy.

Keywords: bioconjugation; functionalization; gene delivery; gold nanoparticles.

*Corresponding author: Narayan Bhattarai, Department of Chemical, Biological and Bioengineering, and Engineering Research Center- Revolutionized Metallic Biomaterials, North Carolina A&T State University, McNair 326, Greensboro, NC 27411, USA, e-mail: [email protected] Bahadur K.C. and Bindu Thapa: Department of Drug Discovery and Biomedical Sciences, South Carolina College of Pharmacy, University of South Carolina, 715 Sumter Street, Columbia, SC 29208, USA

1 IntroductionGene delivery has been utilized as a promising technology for the treatment of inherited and acquired diseases result-ing from abnormal gene expression. It incorporates the delivery of exogenous genetic material to target cells using specific vectors as the delivery of genetic materials on its own suffers from unexpected degradation in the physi-ological environment. Presently, two vectors, viral and nonviral, are used in research and clinical application. In spite of low efficiency, nonviral vectors have been widely used in a wide range of gene delivery application due to their flexible and facile chemistry, cost effectiveness,

and superior safety profiles. In the past decade, nonviral vectors that include polymeric systems (i.e., dendrimers, micelles, and nanoparticles), liposomes, ceramic parti-cles, carbon nanotubes, and metal nanoparticles (i.e., nanorods and nanoparticles) have been widely used as carrier systems [1–3]. Currently, there are six cancer clini-cal trials underway using nanoparticle-based siRNA deliv-ery, but all the nanoparticle-formulated siRNA delivery systems for cancer therapy that are currently in clinical trials are based on polymers or liposomes [3]. However, the efficiency of these constructs always remains insig-nificant for clinical application probably due to lack of enough loading efficiency, less biocompatibility and extracellular stability, insufficient intracellular release, and nuclear delivery, etc.

Among the long list of carriers systems, gold nano-particles (AuNPs) are the leading metal nanoparticles for gene delivery applications along with other biomedical applications, such as diagnostic and therapeutic deliv-ery vehicles [4]. Recently, AuNPs have been utilized in these applications as a result of being stable, uniform, and biocompatible metal nanoparticles with unique electronic structures; size-related intensity display; and highly tunable electronic, magnetic, and optoelectronic properties [5]. Unlike polymeric nanoparticles, the opto-electronic properties of AuNPs reflect a typical electronic band called “surface plasmon resonance (SPR)”, under quantum mechanics, and strongly depend on particle size, shape, interparticle distance, and protecting shell [4, 6]. Furthermore, a slight deviation in the nanoparticles’ par-ticle size can create a radical change in its properties and reflected through SPR. Figure 1 shows the facial synthesis of functionalized AuNPs for a wide range of applications in biomedical research including sensing, imaging, and treatment of inherent and acquired diseases [5, 7]. The promising application of AuNPs is due to its straightfor-ward formulation chemistry, which generates size com-mensurate with multiple biomolecules that enables the integration into the biological system [8]. The soft surface chemistry of AuNPs enables to tailor with various biomol-ecules/ligand via thiol-chemistry, which is impossible in

270 R. Bahadur K.C. et al.: Gold nanoparticle-based gene delivery

other metal nanoparticles (e.g., magnetic nanoparticles). The most important feature of AuNPs is its metallic core building block, which provides a solid support to thera-peutics materials and remains stable even after infinite dilutions, which is impossible in regular polymeric nano-carriers. The high surface-to-volume ratio has increased the performance of AuNPs in delivery applications com-pared to polymeric nanoparticles as it enables them to maximize the payload/carrier ratio exponentially [8].

In gene delivery, AuNPs are used as potential plat-form to load nucleic acids either by direct conjugation or by adsorbing via pregrafted cationic polymeric stabilizers. The unique chemical properties of AuNPs enable for the functionalization with a wide range of polymeric or bio-logical ligands and leads to the formation of stable and biologically friendly, multifunctional, colloidal systems. It includes oligonucleotide-modified AuNPs, cationic quaternary ammonium or branched PEI-functionalized AuNPs, cationic lipid bilayer-coated AuNPs, etc. [9–11]. Thiol end-capped nucleotide molecules are directly grafted onto AuNPs, and grafting density is controlled by varying of end-cap functionality (i.e., monothiol-, tetrathiol-, etc.) [9]. This is an excellent approach to increase the loading amount of nucleic acid unlike polymeric carriers where area-to-volume ratio does not exist. Cationic polymers are routinely grafted onto AuNPs to increase the exposure toward anionic genetic materials. Electrostatic interac-tions between cationic amines of polymers and anionic phosphate groups of genetic material form condensed complexes that protect the payload from enzymatic and nonenzymatic degradation, enhance the cellular uptake via the interaction with anionic cell surface (proteogly-cans), and increase the half-life in the cytoplasm [12, 13]. Unlike polymeric and liposomal carriers, these construct dissemble and release of payload promptly as payloads are loosely adsorbed on the surface. Bonoiu et al. have reported an excellent feature of gold nanorod for siRNA

delivery to target cells or tissues. Gold nanorod complexed with siRNA molecules significantly reduces the expression of key proteins (DARPP-32, extracellular signal-regulated kinase (ERK), and protein phosphate 1 (pp-1) in dopa-minergic signaling pathway in the brain for the therapy of drug addiction [14]. Recently, a novel approach known as “layer-by-layer assembly” has been developed in AuNP-based gene delivery to enhance the efficacy by prolonging the release kinetics of the payload [15]. These assemblies are constructed using stimuli-responsive cationic poly-mers and counter polyelectrolyte nucleic acids. Intracel-lular stimuli-triggered polymer degradation leads to the release of the sandwiched payload molecules that help to maintain the therapeutic window and finally enhance the efficacy of the system [16].

Like other nanoparticles, AuNPs display some typical characteristic features that lie in the interface of molecu-lar and bulk materials [5]. These nanoparticles display polydispersity across a given population, and therefore, it is a challenging task to formulate stable and monodis-persed nanoparticles, which possess well-defined sur-faces and morphology, due to the use of a strong reducing agent, which can alter the nucleation and growth at the atomic level [17]. Stability of AuNPs is controlled by graft-ing stabilizer molecules that range from small moieties to large polymers and biomolecules [5]. The efficiency of the grafting reaction may not be well controlled, and the reaction stoichiometry may not translate into nanoparti-cle conjugation as AuNPs are small enough to undergo fre-quent diffusion. Therefore, it will be a challenging issue to have the proper amount of nucleotides molecules in the nucleotide-AuNP complexes to rule out appropriate effi-ciency. In the last few decades, there have been significant research studies in the formulation of target-specific gene delivery vehicles by grafting targeting moieties via stabi-lizer ligands of AuNPs or using crosslinkers [7, 18, 19]. This strategy enhances the efficiency through biorecognition,

Hydrodynamicdiameter (D)=10–20 nm

AuNPs

D

d

Solid diameter (d)=2–10 nm

ApplicationsFormulation Engineered Au nanoparticles

HAuCl4

SH

+

1. Cellular therapy

Labeling

Targeting

2. Tissue repair

3. Drug delivery

4. Gene delivery

5. Sensing and imaging

6. Catalysis

Figure 1 Schematic representation of AuNP formulation and applications.

R. Bahadur K.C. et al.: Gold nanoparticle-based gene delivery 271

but it has equal probability to shield the optoelectronic properties of metallic core due to the generation of a mul-tilayered complex [(core/shell)n] structure where the outer layers protect and insulate the inner layers. Similarly, biofunctionalization of AuNPs is a tough chemical under-taking due to their small size and the conjugation effi-ciency generally being insignificant as molecular crowing squeezes the reactive functionalities into a narrow curva-ture and decreases the exposure to the chemical reaction [20, 21]. Furthermore, the charge density, electrostatic repulsion, nearest-neighbor interactions, and loss of col-loidal stability during reaction may result in low yield of conjugation [22]. Effective purification of bioconjugated AuNPs is another complicated issue as unbounded bio-molecules primarily compete to bind with cell recep-tors and ultimately hinder the cellular communication and uptake of nanoparticles. During in vivo application, functionalized nanoparticles are exposed to all kinds of biomolecules, and opsonization triggers their aggrega-tion and decreases their half-life. However, continuous effort and advancement in bioconjugate chemistry prom-ises to address the genuine issues and develop AuNPs as a generic multifunctional nano-construct for effective cancer gene therapy. The ideal features that ideal AuNPs, as a potential nanocarrier, should be composed of are, (i) control over the size and size distribution, (ii) control over the grafting efficiency by varying the functionalities of grafting moieties, (iii) control over the biomarker grafting to enhance cellular susceptibility via biorecognition, and (iv) control over stimuli sensitivity by generating labile linkages (e.g., -S-S-) that undergo intracellular cleavage and leads to prompt disassembly of carrier systems in the cytosolic environment. The overall goal of engineering AuNPs is to overcome the major bottlenecks of gene deliv-ery vehicles such as condensation, affinity toward cell surface, escape from endosomal-lysosomal network, cyto-plasmic migration, nuclear uptake, and de-condensation of DNA necessary for transcriptional activity [2, 22, 23]. In this review, we will deal with the fundamental synthetic routes of AuNPs, strategies, and major achievements in cancer gene therapy.

2 Rationale for the use of AuNPs in gene delivery

Gene delivery using AuNPs was accompanied in the early 1990s via particle bombardment where DNA-coated AuNPs were introduced into target cell using physical forces [24, 25]. Efficiency of this method was appreciable,

but it was limited only to the peripheral organs. Thereaf-ter, researchers begin to prepare AuNPs using different stabilizers that were introduced into cells during regular cell functioning (e.g., endocytosis) as the size of these nanoparticles was small enough for cellular uptake. Size and size distribution of AuNPs often varies with the content of stabilizer, and it can be monitored through UV via absorption spectroscopy, which reflects variable surface plasmon resonances. Optical properties of AuNPs enable the quantitative evaluation of intracellular fate of AuNPs via transmission electron microscopy (TEM) [26, 27]. As an example, Elbakery et al. observed AuNPs/siRNA complexes in endosomal compartment of CHO-K1 cells through TEM after 6 h of treatment [28]. Recently, AuNP-based gene delivery has been immersed as an effective tool to distinguish the efficacy of carriers based on their intracellular fate via TEM [27].

3 Synthesis of gold nanoparticlesConventionally, two methods of preparation have been practiced: one-phase and two-phase synthesis [7, 29]. The one-phase synthesis method was introduced in 1951 by Turkevitch, where AuNPs were prepared by reducing chloroaurate salt (HAuCl4) by citrate acid in an aqueous medium. This method has greatly impacted the prepara-tion of biomedical-grade AuNPs in the range of 20 nm (Figure 2A) [30]. There is another popular method to control the size and size distribution of AuNPs by using sodium 3-mercaptopropionate as a costabilizer [31]. Brust-Schiffrin published a most relevant method to syn-thesize AuNPs in 1994 with the inspiration of Faraday’s two-phase system using thiol-capped ligands [32]. In this method, the aqueous chloroaurate ions (AuCl4

-) are trans-ferred to organic phase (toluene) using phase-transfer agent (tetraoctylammonium bromide) and then reduced by sodium borohydride (NaBH4) in the presence of a stabi-lizer (dodecanethiol), which generates AuNPs of 1.5–5 nm diameter (Figure 2B) [33]. For the first time, this method explored the stoichiometry of thiol-gold interaction and its merit in stability, handling, characterization, and surface functionalization of AuNPs. These nanoparticles can be further modified with different biomolecules by ligand-exchange reaction, which allows tailoring multiple functionalities. Recently, the Brust-Schiffrin method has been modified in different ways to increase application sensitivity by controlling shape, size, size distribution, and functionalities [17, 34, 35]. AuNP colloidal suspen-sion, while refluxed at solvent boiling temperature with


surface-active ligands (alkylthiols, -amines, -silanes, -phosphines, -halides, etc.), leads to the formation of nearly monodispersed nanoparticles, and the approach is now called “digestive ripening” [36]. In some studies, UV irradiation is frequently employed to get more precise AuNPs by avoiding the use of strong reducing agents. Here, HAuCl4 solution is irradiated with UV-vis, and the slow appearance of pink coloration indicates the forma-tion of AuNPs [37].

4 Functionalization of AuNPs for gene delivery

AuNPs have been employed as a common platform to con-struct nonviral vectors in gene delivery. A summary of the commonly used AuNP constructs as nonviral vectors and their unique features are highlighted (Figure 3). These constructs are composed of a monolayer of genetic mate-rials/or stabilizer molecules generated either by covalent bonding or by electrostatic interaction. Ideal covalent bonding is the gold-thiol bonding, which is developed by the soft characters of gold and sulfur atoms.

4.1 Nucleotide monolayer

The nucleotide monolayer is generated on the AuNPs by electrostatic interaction or by grafting thiol-capped genetic materials via gold-thiol bonding. The electro-static interaction is built between gold and polyden-tate phosphate groups of nucleotide molecules. This bonding is generated via a substitution reaction facili-tated by negatively charged phosphate ions, which has strong bonding energy with gold than carboxylate ions of citrate molecules [38]. Gold-thiol bonding is a simple and straightforward method due to the soft character of

gold and sulfur. It has fundamental merits in formula-tion and application due to its facile chemistry and redox sensitivity [39]. They have enough stability in a physi-ological environment (pH 7.4) but undergo reductive cleavage in an intracellular environment and promptly release the payload [39, 40]. Chemical conjugation of oli-gonucleotides onto AuNPs increases its cellular stability due to elevated steric inhibition of nuclease digestion, which is a likely cause of tight packing [41]. The increased resistance of nuclease digestion enhances the half-life of nucleotide molecules, thereby, increasing the delivery efficacy of the carrier [42]. Cellular uptake of nucleotide-grafted AuNPs primarily occurs through endocytosis and is proportional to grafting density, which is controlled by varying the functionality (e.g., -SH) of nucleotide mol-ecules [9]. Generally, higher grafting density elevates the protein adsorption and facilitates cellular uptake of nanoparticles [43].

Chemical modification generally decreases the phar-macokinetics of therapeutic agents, but end capping and grafting of nucleotides onto the AuNPs is friendly in all aspects. Mirkin et al. grafted alkylthiol-capped oligonu-cleotides onto AuNPs (13 nm) via gold-thiol bonding and evaluated the activity by binding complementary nucleic acids [44]. The activity was completely preserved, and the formulation displays remarkable reversibility at the inter-face of thermal transition temperature [45]. Rosi et al., for the first time, demonstrated the relevance of oligonucleo-tide-conjugated AuNPs in gene delivery [9]. Antisense oli-gonucleotides were grafted onto AuNPs in two densities (45–50 or 110–120 strands/particles) using tetrathiol- and monothiol-modified nucleotides. The stability of AuNPs in nuclease digestion and cellular uptake was remarkably higher (∼99%) than the control groups regardless of the types of nanoparticles. However, target gene silencing was dependent on the types of nanoparticles; particles with higher oligonucleotide density (antisense particle B) display better EGFP silencing (20%) than the particles

HAuCl4

TOAB

SH

NaBH4 SHAuS Au

S AuS

SH

HAuCl4 + Au

Citrate molecules

O O–

O

OHO

O––O

A

B

Figure 2 Schematic representation of AuNP formulation by (A) one phase system by citrate reduction (B) two phase system reduction followed by stabilization and functionalization via ligand exchange reaction, Brist-Schiffrin method.


with lower density (antisense particle A), and the overall efficiency was significantly higher than commercial trans-fecting agents, LipofectamineTM 2000 and cytofectin. In some studies, siRNA has been modified with PEG via disulfide bonding and then grafted onto AuNPs [46]. This strategy enhances the stability of AuNPs in physiological

systems as well as generates a redox-sensitive protective shell around nanoparticles. Cellular uptake and target gene silencing efficacy of PEG-modified siRNA-complexed AuNPs (AuNPs/siRNA-PEG) was higher than regular AuNPs/siRNA complexes in human prostate carcinoma cells as the AuNPs/siRNA promptly get aggregated.

Stabilizers AuNPs construct ReferencesUnique features

Higher affinity constant for complementarynucleic acids, less susceptible to degradation bynuclease activity, less toxic, higher cellularuptake

[9, 44]

Higher binding efficiency, higher stability inphysiological environment, higher cellular uptakeand teansfection efficiency, less cellular toxic

[51, 61, 63]

Higher stability of lipid-DNA complexes, highercellular uptake and transfection efficiency

[10, 53, 75]

Higher loading content, prolong release kinetics,higher stability of encapsulated nucleotidemolecules, higher efficiency

[15, 78]

Higher stability in physiological condition,enhanced bio-distribution, better serumcompatibility and less toxic

[46]

Increase the stability against enzymatic/non-enzymatic digestion, less cellular toxicity, higherbio-distribution

[27, 73]

Higher stability in physiological condition,stimuli responsive, higher loading efficiency andhigher cellular uptake

[51, 74]

Figure 3 AuNP construct for gene delivery using different stabilizers. The assembled platforms incorporate genetic materials either by covalent bonding or by electrostatic interaction and display unique characteristic features, which enable them as a potential carrier for gene delivery.


4.2 Cationic polymer monolayer

Cationic polymer/molecules are grafted onto AuNPs to generate cationic monolayers, which provides enough exposure to nucleic acids (DNA/RNA) and enables elec-trostatic adsorption. This interaction forms tightly packed complexes, which inhibit the enzymatic/nonenzymatic degradation of nucleotide molecules and increase their half-life in physiological environments [16, 28, 47–49]. Common cationic stabilizers exploited in AuNP-based gene delivery are cationic amino acids, polyethyleneimine (PEI), chitosan, quaternary ammonium chains, 2-ami-noethanethiol, and cationic lipids [50–54]. Size and size distribution of the nanoparticles generally increases with the content of the stabilizer and display significant impact on transfection efficiency; therefore, the smaller the sizes, the better the efficiency [55].

Higher molecular weight cationic polymers inher-ently have enough capacity to complex and condense nucleic acids into the polyionic nanoparticles required for transport into target cells. However, there has been serious concern regarding cellular uptake, endosomal stability, lysosomal release, and intolerable toxicity caused by aggregation and adherence on the cell surface, which leads to significant necrosis [56, 57]. To this end, these polymers are modified to second-generation formu-lation by grafting onto AuNPs, which enhance cellular

communication within safe toxicity profile, while retain-ing the potent transfection ability [58]. Chitosan, PEI, and PLL are routinely grafted onto AuNPs through the lone pair electrons of the nitrogen atoms, which generate a tight binding with AuNPs [59]. In some studies, cationic polymers are chemically modified to generate reliable functionality (e.g., -SH, hydrophobic moieties) to anchor onto AuNPs (Figure 4).

Low molecular weight chitosan has been modified to generate thiol- or aliphatic lipid functionality and grafted onto AuNPs [60]. The ideal nanoparticles were formulated by anchoring onto AuNPs either through gold-thiol bonding via a ligand exchange reaction or through interdigitated bilayer formation with cetyltrimethylam-monium bromide. Chitosan (Mw 6 kDa)-grafted AuNPs (Chito6-AuNPs) have been reported to have significant DNA delivery efficiency on in vitro/in vivo model [61]. Intramuscular immunization of Chito6-AuNPs in BALB/c mice induced an enhanced serum antibody response, 10 times more potent than a naked DNA vaccine. In spite of potent efficiency, high molecular weight chitosan has some issues such as, solubility and higher viscosity that leads to limited clinical application. To resolve this issue, aliphatic lipids are routinely grafted onto high molecular weight chitosan and then grafted onto AuNPs. Bhattarai et al. reported a promising result on in vitro/in vivo gene delivery using hydrophobically modified chitosan-grafted

AuAu Au

Au

Figure 4 Schematic representation for chemical modification of cationic polymers (e.g., chitosan) and AuNP formulation.


AuNPs [62]. Hydrophobic modification of high molecu-lar weight chitosan (viscosity average molecular weight, Mv = 2.1 × 105, degree of deacetylation 78%) with aliphatic lipids increases its solubility in aqueous mediums, which can be further increased by grafting onto AuNPs. The effi-ciency of plain chitosan or modified chitosan and plain chitosan-stabilized AuNPs was negligible, but the effi-ciency of modified chitosan-stabilized AuNPs was dra-matically higher in breast cancer cell lines. The efficiency in in vivo studies was remarkably higher than commer-cially available transfecting agents Lipofectin™ 2000.

PEI is used as a gold standard nonviral vector for in vitro and in vivo gene delivery. AuNPs are functional-ized with PEI to import its beneficial features required for gene delivery [52, 63]. These are strong cationic poly-mers and display enough buffering effect, in a wide range of pH, which facilitate endosomal escape of the carriers via a “proton-sponge” effect. Thomas et al. has prepared low molecular weight (MW 2 kDa) PEI (PEI2)-grafted gold nanoparticles (PEI-AuNPs) and employed pDNA delivery to monkey kidney (COS-7) cells [51]. Transfection effi-ciency of nanoparticles was slightly increased (25%) com-pared to unmodified PEI2. Interestingly, the efficiency was increased (50%) by the addition of N-dodecyl-PEI2. Here, the synergistic effect of N-dodecyl-PEI2 was attrib-uted to hydrophobicity, which generally enhances the cellular communication. The metabolic activity of cells treated with PEI-AuNPs/pDNA was still higher (∼80%), but it was decreased (∼70%) on ternary complexes [51]. In some studies, high molecular weight (MW 25 kDa) PEIs (PEI25) are used to stabilize AuNPs (PEI-AuNPs) as the tertiary amine of these polymer act as a reductant as well as a stabilizer [64, 65]. Song et al. formulated monodis-persed PEI25-AuNPs by incubating aqueous HAuCl4 with PEI25 at room temperature and complexing with siRNA. The in vitro study reported a remarkable cellular uptake of PEI25-AuNPs/siRNA in MDA-MB435 cells. These com-plexes display significant knockdown of GFP protein and endogenous cell cycle kinase (PLK1) expression and induce enhanced cell apoptosis expression [58]. In some studies, intermediate molecules are employed to reduce and graft AuNPs onto polymeric materials. This strategy basically improves the size distribution and functionaliza-tion susceptibility of nanoparticles. Catechol is a common hydrophobic molecule that reduces counter molecules by donating lone pair electrons and converting itself into a highly reactive quinone form, which has high affinity toward amines and thiols via Michael-type addition reac-tion [66, 67]. Lee et al. reported facile synthesis of AuNPs using catechol and then functionalization with PEI25/PEG [68]. These nanoparticles exhibited low toxicity

and display excellent GFP gene silencing in MAD-MB435 human breast cancer cell lines.

Amino acids and polypeptides are grafted onto AuNPs to generate bio-inspired features other than neutral hydro-philic polymers (i.e., zwitterions enhance solubility and binding efficiency). High charge density of lysine and first-generation lysine-dendron-functionalized AuNPs enables the formation of compact complexes with pDNA as the 3D spherical morphology of the stabilizer allows for more efficient interaction [54, 69]. Lysine first-generation den-dron-capped AuNPs display remarkable redox sensitivity and 28-fold superior transfection efficiency in monkey kidney cells (Cos-1) compared to poly-l-lysine (PLL). Many studies reported that PLL is used as a capping and reduc-ing agent to prepare PLL-AuNPs like PEI25 polymers [64, 65, 70]. This method generates monodispersed PLL-AuNP nanoparticles with narrow size distribution and display significant pDNA binding efficacy. Transfection efficiency of PLL-AuNPs/pDNA complexes in NIH-3T3 fibroblast cells is significant and always proportionate to the molecu-lar weight of PLL. AuNPs capped with higher molecular weight (MW 30–70 kDa) PLL display superior transfection that was comparable to PEI25/pDNA. Furthermore, cellu-lar uptake and transfection activity of PLL-AuNPs/pDNA complexes can be maintained almost 2 weeks without compromising cell viability.

4.3 Nucleotides and polymer-mixed monolayer

PEG and cyclodextrin (CD) are common polymers grafted into nanocarriers to generate a biocompatible hydro-philic corona. This hydrophilic corona decreases the adsorption of plasma proteins and cellular elements in blood and increases navigation time in the circula-tory system [71, 72]. PEG is used to stabilize AuNPs as well as an anchoring spacer, although there remains a serious concern about its steric hindrance. Kawano et al. had formulated mixed monolayer cationic AuNPs using mPEG-SH and 2-aminoethanethiol and demonstrated an excellent bio-distribution in mice model with signifi-cant gene expression [73]. Oishil et al. stabilized AuNPs by poly(ethylene glycol)-b-poly(2(N-N-dimethylamino)ethyl methacrylate copolymer and incubated with HS-siRNA to generate a mixed monolayer as well as to physi-cally complex with siRNA, which was then evaluated for silencing of luciferase expression in human hepatoma cell (HuH-7) [40]. AuNPs with mixed monolayer dis-played superior silencing efficacy (65%) compared to nanoparticles with physically adsorbed siRNA (25%).


The higher efficiency was attributed by the reductive cleavage of gold-thiol bonding as observed elsewhere [39]. Recently, Conde et al. designed multifunctional AuNPs using heterofunctional PEGs that were anchored with targeting ligands [27]. siRNA was grafted cova-lently (NP-cov) or electrostatically (NP-ion), and then the silencing efficiency was evaluated in in vitro as well as in vivo models. The nanocomplexes display supe-rior stability under reasonable size distribution due to the presence of hydrophilic functionality. The overall finding suggested that the efficiency of NP-cov was supe-rior in both models. Recently, significant progress has been made in designing redox-sensitive AuNPs for gene delivery. These nanoparticles are prepared by grafting stabilizers/nucleic acids via disulfide bonding. Lee et al. prepared PEG (MW 1.0 kDa)-stabilized AuNPs, grafted the siRNA via disulfide bonding, and finally evaluated the siRNA delivery efficiency to luciferase-expressing HeLa cells [74]. siRNA-grafted nanoparticles were coated with biodegradable cationic polymers [poly(β-amino ester)s (PBAEs)] to enhance cellular uptake and endo-somal escape. The coated nanoparticles display remark-able knockdown of target gene expression, while the efficiency of bare nanoparticles was negligible.

A mixed monolayer, composed of neutral and cati-onic hydrophobic chains, is generated around AuNPs using the Murray displacement method to increase the exposure of nanoparticles toward biomacromolecules. A trimethylamonuim capped aliphatic chain is a common cationic molecule used to generate mixed monolayer-protected gold clusters (MMPCs) [39, 49]. The mixed mono layer is composed with hydrophobic alkane and cationic trimethylammonium alkane chains of differ-ent lengths. The self-assembled monolayer provides a highly organized cationic surface, which is selectively susceptible to nucleotide molecules. It increases binding affinity, stability against DNAse I digestion in physiologi-cal environment, and physical/chemical exposure [48]. Sandhu et al. reported significant transfection efficiency of MMPCs in mammalian cells (293T cells); however, the efficiency primarily depends on different variables such as pDNA/nanoparticle ratio, number of charged substit-uents in the monolayer core, and hydrophobic packing [50].

4.4 Cationic lipid assembly of AuNPs

Cationic lipids have been used as prominent nonviral vectors for gene delivery during the last few decades. They form complexes (lipoplexes) with nucleic acids

(DNA/RNA) through cationic bonding and display supe-rior cellular communication. However, frequent applica-tion of these carriers in clinical settings is limited due to intolerable toxicity and improper solubility in aqueous medium at physiological conditions. There have been significant studies to synergize the beneficial effect of cationic lipids and AuNPs for gene delivery by formulat-ing a hybrid construct. Stability of lipid-DNA complexes significantly increases, while lipid molecules are coated onto AuNPs, and these hybrid complexes display better pDNA delivery efficiency than the parent lipid (dime-thyldiocatadecylammonium bromide, DODAB) [75]. On the contrary, some studies dealt with the formulation of cationic lipid-coated AuNPs-pDNA hybrid complexes by electrostatic interaction of counter stabilizers [76]. It begins with replacing citrate molecules of AuNPs by pDNA molecules and then adsorbing cationic lipid molecules, which leads to the formation of lipid layers around pDNA-AuNPs complexes. The hybrid nanopar-ticles display superior transfection efficiency in differ-ent human cancer cells lines (A549 cells, lung cancer cells, HeLa cells, cervical cancer cells) compared to the parent molecules (liposome, AuNPs) under safe toxicity profiles which is significantly higher (94–98%) than the Lipofectamine™ 2000. The higher efficiency of hybrid nanoparticles is because it is less likely for pDNA to be degraded or detached inside or outside the cells conse-quently delivered to the nucleus without inducing any toxicity [76]. Lipid-AuNPs hybrid nanoparticles are also formulated by emulsification where nanoparticles were encapsulated into liposomal vesicles [10, 53]. Kong et al. has prepared these hybrid constructs by the emulsifica-tion of hydrophobic dodecanethiol-capped AuNPs and three different lipid components: DC-Chol, DOPE, and Chol [10]. Emulsification assembles amphiphilic lipid building blocks around AuNPs and exposes the positively charged shell, which is expected to effectively condense nucleic acids. The hybrid construct exhibited significant cellular uptake and GFP gene silencing efficacy in human cancer cells (MAD-MB-435, A549 cells) with remarkably low cytotoxicity.

4.5 Layer-by-layer assembly of AuNPs

Layer-by-layer (LbL) fabrication is a versatile assembly technology to generate multiple thin films on flat solid sur-faces, microparticles, and nanoparticles via electrostatic interaction of oppositely charged polyelectrolytes [15]. In AuNPs, multiple layers of genetic materials are sand-wiched between biodegradable polymers, and polymer


degradation triggers the release kinetics of genetic mate-rials, which ultimately prolonged the efficacy of the system. Lee et al. prepared a protease-degradable AuNPs assembled with PLL and siRNA and reported silencing efficacy based on siRNA loading content and polymer degradation time [16]. Silencing of luciferase gene in MDA-MB231-luc2 cells was gradually increased with time and implying the sustained release of siRNA. Generally, toxicity of AuNPs is the cause of surface molecular com-position, and higher molecular weight polycations are critical in this regard. However, AuNPs assembled with PLL and siRNA displayed insignificant toxicity in MDA-MB231-luc2 cells compared to LipofectamineTM 2000 [16]. LbL technology is also a straightforward way to tune surface properties of nanoparticles and directly impacts the efficacy of carrier systems [28]. The LbL technique is also used to neutralize the surface charge of nanocarri-ers as strong cationic charges induce nonspecific adsorp-tion of blood serum components as well as binding to negatively charged cell membrane resulting in cell nar-cosis. This idea mimics the technique of tertiary systems, where the cationic charge of the nanocarrier is neutral-ized via adsorbing polyanions [77]. Lee et al. reported an approach to formulate a nanocarrier for target-specific siRNA delivery by LbL assembly of cysteamine-capped AuNPs using counter polymers PEI and hyaluronic acid (HA) (AuNPs-CM/siRNA/PEI/HA) [78]. HA adsorption generally increases the size and decreases the surface charge of nanoparticles. Consequently, it minimizes the adsorption of serum components thereby preventing the aggregation in cell culture medium. With negligi-ble cytotoxicity, AuNP-CM/siRNA/PEI/HA nanocarriers display significant luciferase gene silencing (70–80%) in B16F1 cells even at high serum concentration (50%). The higher efficacy of the nanoparticles was primarily due to enhanced serum stability and cellular uptake through HA receptor-mediated endocytosis.

5 Conclusion and remarksConsiderable improvement has been developed over the last few years in the design of gold nanoparticles for drug/gene delivery. However, the efficacy has not improved enough to overcome counter carriers (e.g., viral vectors, lipoplexes, and polyplexes). The standard requirement for clinical applications has not been reached. This is primarily due to the lack of efficiency and specificity. The advancement of bioconjugation chemistry, molecu-lar biology, and continuous effort of study promises to develop multifunctional gold nanoparticles, which will lead biomedical research to a new avenue of diagnosis and therapy. Gene delivery, a multistep process, should have research focused on the design of appropriate carries that can overcome each hurdle of the delivery trajectory. An ideal carrier can be designed by tailoring targeting ligands and stimuli-responsive segments onto a physi-ological friendly dimension. Currently, the prevailing impression in clinical research is that the expression of a single transgene is unlikely to be enough for tumor treat-ment. Similarly, characterization of systemic performance of AuNP in vivo studies, including immune stimulation, cytotoxicity, pharmacokinetics, biodistribution, etc., is essential to advance future nanomedicine. Therefore, it is critical to deliver multiple therapeutic agents using a single platform “multitarget smart carriers”, and gold nanoparticles deserve to be tailored in this dynamics.

Acknowledgments: N. Bhattarai would like to acknowledge the National Science Foundation (NSF)-Nanotechnology Undergraduate Education (NUE-1242139) and NSF-Engi-neering Research Center for Revolutionizing Metallic Bio-materials (ERC-0812348) for their financial support.

Received September 11, 2013; accepted October 23, 2013; previously published online December 10, 2013

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Remant Bahadur K.C. is a postdoctoral fellow of the Department of Drug Discovery and Biomedical Sciences, South Carolina College of Pharmacy, University of South Carolina, Columbia, SC, USA. He received a Master’s degree in Organic Chemistry (MSc) from Tribhuvan University, Nepal and a PhD in Bionanosystem Engineering from Chonbuk National University, South Korea. His research interest includes the design of novel biomaterials for the development of drug/gene delivery carriers. Currently, his research is focused on the synthesis of functional polymers to design smart nanocarriers for nonviral gene therapy.

Bindu Thapa is a research assistant at the Department of Drug Discovery and Biomedical Sciences, South Carolina College of Pharmacy, University of South Carolina, Columbia, SC, USA. She received a Master’s degree in Pharmaceutical Science (M. Pharm.) from Pokhara University, Nepal. Her research interest includes pharmaceutics and biopharmaceutics. Currently, her research is focused in bioconjugation, nanoformulation, and cancer therapy.

Narayan Bhattarai received a MS in Physical Chemistry from Tribhuvan University, Nepal, and a PhD in Materials Engineering from Chonbuk National University, South Korea (2000–2003). He obtained his postdoctoral training from the University of Washington (2003–2008) and was appointed as an instructor at the University of Washington, Department of Materials Science and Engineering (2008–2010). Currently, he serves as assistant professor of Bioengineering in North Carolina A&T State University (NCAT). He is principle investigator (PI) of the NSF-funded Nanotechnology for Undergraduate Education (NUE) at NCAT. He is also affiliated as an investigator with NSF’s Engineering Research Center for Revolutionizing Metallic Biomaterials (ERC-RMB) at NCAT. His research interest includes synthesis and modifications of biomedical polymers and composites; surface engineering of metallic and ceramic implant materials; biomimetic nanofibers and 3D scaffolds for tissue regeneration, cellular differentiation, and cancer treatment; engineered nanoparticles for therapeutic delivery and imaging.

remant bahadur k.c., bindu thapa and narayan bhattarai

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